Morphological evidence of telocytes in human synovium

Abstract

A new cell type named telocyte (i.e. cell with distinctive prolongations called telopodes) has recently been identified in the stroma of various organs in humans. However, no study has yet reported the existence of telocytes in the synovial membrane of diarthrodial joints. This work was therefore undertaken to search for telocytes in the normal human synovium using transmission electron microscopy, immunohistochemistry and immunofluorescence. Ultrastructural analyses demonstrated the presence of numerous spindle-shaped telocytes in the whole synovial sublining layer. Synovial telocytes exhibited very long and thin moniliform telopodes and were particularly concentrated at the boundary between the lining and sublining layers and around blood vessels. Light microscopy confirmed the presence of CD34-positive telocytes in the aforementioned locations. Moreover, synovial telocytes coexpressed CD34 and platelet-derived growth factor receptor α. Double immunostaining further allowed to unequivocally differentiate synovial telocytes (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). The in vitro examination of fibroblast-like synoviocyte primary cultures revealed the coexistence of different cell types, including CD34-positive telocytes projecting typical moniliform telopodes. In conclusion, our work provides the first evidence that telocytes do exist in the human synovium and lays the groundwork for future studies on synovial telocytes in a variety of degenerative and destructive joint diseases.

Figure 1

Morphology of human synovium (lining and sublining layers) under light and transmission electron microscopy. (a,b) Representative photomicrographs of toluidine blue-stained synovial semithin sections. Arrowheads in (a,b) point to the synovial lining layer, while asterisks in (b) denote blood vessels in the synovial sublining layer. (b) Stromal cells exhibiting a spindle-shaped or piriform cell body with a large nucleus and very small amount of cytoplasm, and long and thin cytoplasmic processes are observed at the boundary between the synovial lining and sublining layers (arrow; shown at higher magnification in the inset). (c,d) Representative transmission electron microscopy photomicrographs of synovial ultrathin sections stained with uranyl acetate and bismuth subnitrate solutions. Telocytes (TC) and telopodes (Tp) have been digitally colored in blue. The ultrastructural traits of telocytes are: i) a spindle-shaped or piriform cell body with a relatively large euchromatic nucleus surrounded by a thin cytoplasmic layer containing few mitochondria, scarce cisternae of endoplasmic reticulum and a small Golgi apparatus, and ii) the presence of telopodes, long cytoplasmic processes with a narrow emergence from the cell body and a moniliform silhouette characterized by the alternation of thin segments (podomers) and expanded parts (podoms). Telocytes and telopodes are present in the synovial sublining layer immediately beneath the type A/macrophage-like synoviocytes (Ma) and the type B/fibroblast-like synoviocytes (Fb) constituting the synovial lining (c,d). The synovial sublining contains either telopode-bearing telocytes or fibroblasts (Fb), these latter displaying an abundant cytoplasm rich in cisternae of rough endoplasmic reticulum, mitochondria and Golgi apparatus, and short and thick processes (c). Scale bar: 50 µm (a,b), 5 µm (c,d).

Figure 2

Morphology of human synovium (sublining layer) under light and transmission electron microscopy. (a,b) Representative photomicrographs of toluidine blue-stained synovial semithin sections. Asterisks denote sublining layer blood vessels. Stromal cells displaying a spindle-shaped or piriform cell body with a large nucleus and very small amount of cytoplasm, and long and thin moniliform cytoplasmic processes are present throughout the synovial sublining, particularly in perivascular locations (arrows; shown at higher magnification in the insets). (c–h) Representative transmission electron microscopy photomicrographs of synovial ultrathin sections stained with uranyl acetate and bismuth subnitrate solutions. Telocytes (TC) and telopodes (Tp) have been digitally colored in blue. Telocytes are ultrastructurally characterized by i) a small cell body mostly occupied by a relatively large nucleus surrounded by scarce cytoplasm containing few mitochondria and cisternae of endoplasmic reticulum and a small Golgi apparatus, and ii) thin moniliform processes (telopodes) with narrow emergence from the cell body and a moniliform aspect (alternating thin segments/podomers and dilated portions/podoms). The nucleus is indented, showing patches of heterochromatin near the nuclear membrane. Podoms accommodate some organelles, such as rough endoplasmic reticulum and mitochondria (c, inset). The ultrastructural traits of telocytes make them easily distinguishable from neighboring fibroblasts (Fb), which exhibit a large body rich in cisternae of rough endoplasmic reticulum and mitochondria, a large Golgi apparatus and short and thick processes (d). Telocytes and telopodes can be observed throughout the whole synovial sublining layer, either in neutral positions surrounded by abundant collagenous extracellular matrix (c,e), or topographically closely related to fibroblasts (d) and blood vessels (BV) (f–h). Note the presence of telopodes delimiting the basal lamina of microvessels (f–h). Scale bar: 50 µm (a,b), 5 µm (c–h).

Figure 3

Representative light and fluorescence microscopy photomicrographs of human synovial sections. (a–c) CD34 immunoperoxidase-based immunohistochemistry with hematoxylin counterstain. (d) Immunofluorescence labeling for CD34 (green) with 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain for nuclei. CD34-positive spindle-shaped cells (telocytes) with long cytoplasmic processes (telopodes) are distributed throughout the whole synovial sublining layer (a–d). Note the presence of numerous CD34-positive stromal cells arranged in multiple parallel rows at the synovial lining-sublining interface (arrowheads in a,b) and surrounding sublining microvessels (arrows in b,c). CD34 immunopositivity is observed also in vascular endothelial cells. At higher magnification, the processes of CD34-positive stromal cells exhibit a moniliform silhouette (d, inset). (e,f) Double immunofluorescence labeling for CD34 (green) and PDGFRα (red) with DAPI (blue) counterstain for nuclei. Telocytes are double positive for CD34 and PDGFRα. (g) Double immunofluorescence labeling for CD34 (green) and CD31 (red) with DAPI (blue) counterstain for nuclei. Telocytes are CD34-positive/CD31-negative, while vascular endothelial cells are CD34-positive/CD31-positive (arrows). Scale bar: 50 µm (a–g).

Figure 4

Representative phase-contrast and fluorescence microscopy photomicrographs of human fibroblast-like synoviocyte primary cultures. (a,b) Phase-contrast microscopy. (c,d) Wheat Germ Agglutinin (WGA; green) immunofluorescence labeling with 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain for nuclei. (a–d) Note the presence of either fibroblasts (Fb) or telocytes (TC), the latter being characterized by a small cell body and very long and thin moniliform processes (telopodes, Tp) often establishing intercellular contacts with fibroblasts or other telocytes (arrowheads). (e,f) Immunofluorescence labeling for CD34 (red) with DAPI (blue) counterstain for nuclei. CD34-positive cells projecting very long and thin moniliform cytoplasmic processes (telopodes, Tp) are unequivocally identifiable as telocytes (TC). The inset in (e) shows a higher magnification of a typical telopode formed by an alternation of thin segments (podomers) and expanded parts (podoms). Note the telopode of a telocyte contacting the cell body of another telocyte (arrowhead in e). Scale bar: 50 µm (a–f).